Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome p450 metabolites of arachidonic acid

Kasem Nithipatikom, Andrew J. Grall, Blythe B. Holmes, David R. Harder, J R Falck, William B. Campbell

Research output: Contribution to journalArticle

103 Scopus citations

Abstract

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.

Original languageEnglish (US)
Pages (from-to)327-336
Number of pages10
JournalAnalytical biochemistry
Volume298
Issue number2
DOIs
StatePublished - Dec 15 2001

Keywords

  • 20-hydroxyeicosatetraenoic acid
  • Arachidonic acid
  • Astrocytes
  • Cytochrome P450
  • Dihydroxyeicosatrienoic acids
  • Endothelial cells
  • Endothelial-derived hyperpolarizing factor (EDHF)
  • Epoxyeicosatrienoic acids
  • LC-MS

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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